Steady-state measurements of lactic acid production in a wild-type and a putative d-lactic acid dehydrogenase-negative mutant of zymomonas mobilis

This work represents a continuation of our investigation into environmental conditions that promote lactic acid synthesis by Zymomonas mobilis. The characteristic near theoretical yield of ethanol from glucose by Z. mobilis can be compromised by the synthesis of d- and l-lactic acid. The production of lactic acid is exacerbated by the following conditions: pH 6.0, yeast extract, and reduced growth rate. At a specific growth rate of 0.048/h, the average yield of dl-lactate from glucose in a yeast extract-based medium at pH 6.0 was 0.15 g/g. This represents a reduction in ethanol yield of about 10% relative to the yield at a growth rate of 0.15/h. Very little lactic acid was produced at pH 5.0 or using a defined salts medium (without yeast extract) Under permissive and comparable culture conditions, a tetracycline-resistant, d-ldh negative mutant produced about 50% less lactic acid than its parent strain Zm ATCC 39676. d-lactic acid was detected in the cell-free spent fermentation medium of the mutant, but this could be owing to the presence of a racemase enzyme. Under the steady-state growth conditions provided by the chemostat, the specific rate of glucose consumption was altered at a constant growth rate of 0.075/h. Shifting from glucose-limited to nitrogen-limited growth, or increasing the temperature, caused an increase in the specific rate of glucose catabolism. There was good correlation between an increase in glycolytic flux and a decrease in lactic acid yield from glucose. This study points to a mechanistic link between the glycolytic flux and the control of end-product glucose metabolism. Implications of reduced glycolytic flux in pentose-fermenting recombinant Z. mobilis strains, relative to increased byproduct synthesis, is discussed.     

A simultaneous sucrose bioconversion into ethanol and levan byZymomonas mobilis

Two biotechnological systems were developed for sucrose conversion into levan and ethanol withZymomonas mobilis, ensuring a 66.7% transfer of substrate carbon in a batch and 61% carbon transfer in a continuous culture. The effect of glucose, ethanol, and medium pH on sucrose conversion byZ. mobilis was studied. The addition of ethanol to the fermentation medium, in the final conc. of 100 g/L, uncoupled levan synthesis from ethanol fermentation. For a continuous culture, the most efficient conversion of substrate carbon into levan was reached at pH 4.8, giving 64.2 g/L levan, with the levan yield of 0.22 g/g and the productivity of 3.2 g/L/h.     

Continuous culture studies of xylose-fermentingZymomonas mobilis

The continuous cofermentation performance of xylose-fermentingZymomonas mobilis at 30°C and pH 5.5 was characterized using a pure-sugar feed solution that contained 8 g/L glucose and 40 g/L xylose. Successful chemostat start up resulted in complete utilization of glucose and greater than 85% utilization of xylose, but was only reproducibly achieved using initial dilution rates at or less than 0.04/h; once initiated, cofermentation could be maintained at dilution rates of 0.04 to 0.10/h. Whereas xylose and cell-mass concentrations increased gradually with increasing dilution rate, ethanol concentrations and ethanol yields on available sugars remained approximately constant at 20–22 g/L and 80–90% of theoretical, respectively. Volumetric and specific ethanol productivities increased linearly with increasing dilution rate, rising from approx 1.0 each (g/L/h or g/g/h) at a dilution rate of 0.04/h to approx 2.0 each (g/L/h or g/g/h) at a dilution rate of 0.10/h. Similarly, specific sugar-utilization rates increased from approx 2.0 g/g/h at dilution rate 0.04/h to approx 3.5 g/g/h at dilution rate of 0.10/h. The estimated values of 0.042 g/g for the maximum Z.mobilis cell-mass yield on substrate and 1.13 g/g/h for the minimum specific substrate utilization rate required for cellular maintenance energy are within the range of values reported in the literature. Results are also presented which suggest that long-term adaptation in continuous culture is a powerful technique for developing strains with higher tolerance to inhibitory hemicellulose hydrolyzates.     

Fermentation performance assessment of a genomically integrated xylose-utilizing recombinant of Zymomonas mobilis 39676

In pH-controlled batch fermentations with pure sugar synthetic hardwood hemicellulose (1% [w/v] glucose and 4% xylose) and corn stover hydrolysate (8% glucose and 3.5% xylose) lacking acetic acid, the xyloseutilizing, tetracycline (Tc)-sensitive, genomically integrated variant of Zymomonas mobilis ATCC 39676 (designated strain C25) exhibited growth and fermentation performance that was inferior to National Renewable Energy Laboratory's first-generation, Tc-resistant, plasmid-bearing Zymomonas recombinants. With C25, xylose fermentation following glucose exhaustion wasmarkellyslower, and the ethanol yield (based on sugars consumed) was lower, owing primarily to an increase in lactic acid formation. There was an apparent increased sensitivity to acetic acid inhibition with C25 compared with recombinants 39676:pZB4L, CP4:pZB5, and ZM4:pZB5. However, strain C25 performed well in continous ferm entation with nutrient-rich synthetic corn stover medium over the dilution range 0.03–0.06/h, with a maximum provess ethanol yield at D=0.03/h of 0.46 g/g and a maximum ethanol productivity of 3 g/(L·h). With 0.35% (w/v) acetic acid in the medium, the process yield at D=0.04/h dropped to 0.32 g/g, and the maximum productivity decreased by 50% to 1.5 g/(L·h). Under the same operating conditions, rec Zm Zm 4:pZB5 performed better; however, the medium contained 20 mg/L of Tc to constantly maintain selective pressure. The absence of any need for antibiotics and antiboitic resistance genes makes the chromosomal integrant C25 more com patible with current regulatory specifications for biocatalysts in large-scale commercial operations.     

Fermentation performance characteristics of a prehydrolyzate-adapted xylose-fermenting recombinant Zymomonas in batch and continuous fermentations

Long-term (149 d) continuous fermentation was used to adapt a xylose-fermenting recombinant Zymomonas mobilis, strain 39676:pZB 4L, to conditioned (overlimed) dilute-acid yellow poplar hemicellulose hydrolyzate (“prehydrolyzate”). An “adapted” variant was isolated from a chemostat operating at a dilution rate of 0.03/h with a 50% (v/v) prehydrolyzate, corn steep liquor, and sugar-supplemented medium, at pH 5.75. The level of xylose and glucose in the medium was kept constant at 4% (w/v) and 0.8% (w/v), respectively. These sugar concentrations reflect the composition of the undiluted hardwood prehydrolyzate. The level of conditioned hardwood prehydrolyzate added to the medium was increased in 5% increments startingata level of 10%. At the upper level of 50% prehydrolyzate, the acetic-acid concentration was about 0.75% (w/v). The adapted variant exhibited improved xylose-fermentation performance in a pure-sugar, synthetic hardwood prehydrolyzate medium containing 4% xylose (w/v), 0.8% (w/v) glucose, and acetic acid in the range 0.4–1.0% (w/v). The ethanol yield was 0.48–0.50 g/g; equivalent to a sugar-to-ethanol conversion efficiency of 94–96% of theoretical maximum. The maximum growth yield and maintenance energy coefficients were 0.033 g dry cell mass (DCM)/g sugars and 0.41 g sugars/g DCM/h, respectively. The results confirm that long-term continuous adaptation is a useful technique for effecting strain improvement with respect to the fermentation of recalcitrant feedstocks.     

The effect of lactic acid on fuel ethanol production byZymomonas

Efficient utilization of the pentosan fraction of hemicellulose from lignocellulosic feedstocks offers an opportunity to increase the yield and to reduce the cost of producing fuel ethanol. During prehydrolysis (acid hydrolysis or autohydrolysis of hemicellulose), acetic acid is formed as a consequence of the deacetylation of the acetylated moiety of hemicellulose. Recombinant Escherichia coli B (ATCC 11303), carrying the plasmid pLO1297 with pyruvate decarboxylase and alcohol dehydrogenase II genes from Zymomonas mobilis (CP4), converts xylose to ethanol with a product yield that approaches theoretical maximum. Although other pentose-utilizing microorganisms are inhibited by acetic acid, the recombinant E. coli displays a high tolerance for acetic acid. In xylose fermentations with a synthetic medium (Luria broth), where the pH was controlled at 7, neither yield nor productivity was affected by the addition of 10.7 g/L acetic acid. Nutrient-supplemented, hardwood (aspen) hemicellulose hydrolysate (40.7 g/L xylose) was completely fermented to ethanol (16.3 g/L) in 98 h. When the acetic acid concentration was reduced from 5.6 to 0.8 g/L, the fermentation time decreased to 58 h. Overliming, with Ca(OH)₂ to pH 10, followed by neutralization to pH 7 with sulfuric acid and removal of insolubles, resulted in a twofold increase in volumetric productivity. The maximum productivity was 0.93 g/L/h. The xylose-to-ethanol conversion efficiency and productivity in Ca(OH)₂-treated hardwood prehydrolysate, fortified with only mineral salts, were 94% and 0.26 g/L/h, respectively. The recombinant E. coli exhibits a xylose-to-ethanol conversion efficiency that is superior to that of other pentose-utilizing yeasts currently being investigated for the production of fuel ethanol from lignocellulosic materials.     

Comparative ethanol productivities of different Zymomonas recombinants fermenting oat hull hydrolysate

Iogen Corporation of Ottawa, Canada, has recently built a 50 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. Iogen has partnered with the University of Toronto to test the C6/C5 cofermentation performance characteristics of National Renewable Energy Laboratory's metabolically engineered Zymomonas mobilis using its biomass hydrolysates. In this study, the biomass feedstock was an agricultural waste, namely oat hulls, which was hydrolyzed in a proprietary two-stage process involving pretreatment with dilute sulfuric acid at 200–250°C, followed by cellulase hydrolysis. The oat hull hydrolysate (OHH) contained glucose, xylose, and arabinose in a mass ratio of about 8:3:0.5. Fermentation media, prepared from diluted hydrolysate, were nutritionally amended with 2.5 mL/L of corn steep liquor (50% solids) and 1.2 g/L of diammonium phosphate. The estimated cost for large-scale ethanol production using this minimal level of nutrient supplementation was 4.4c/gal of ethanol. This work examined the growth and fermentation performance of xyloseutilizing, tetracycline-resistant, plasmid-bearing, patented, recombinant Z. mobilis cultures: CP4:pZB5, ZM4:pZB5, 39676:pZB4L, and a hardwood prehydrolysate-adapted variant of 39676:pZB4L (designated asthe “adapted” strain). In pH-stat batch fermentations with unconditioned OHH containing 6% (w/v) glucose, 3% xylose, and 0.75% acetic acid, rec Zm ZM4:pZB5 gave the best performance with a fermentation time of 30h, followed by CP4:pZB5 at 48h, with corresponding volumetric productivities of 1.4 and 0.89 g/(L·h), respectively. Based on the available glucose and xylose, the process ethanol yield for both strains was 0.47 g/g (92% conversion efficiency). At 48 h, the process yield for rec Zm 39676:pZB4L and the adapted strain was 0.32 and 0.34 g/g, respectively. None of the test strains was able to fermentarabinose. Acetic acid tolerance appeared to be a major determining factor in cofermentation performance.     

Continuous production of lactic acid in a cell recycle reactor

The production of lactic acid from glucose has been demonstrated using a CSTR (continuous stirred-tank reactor) with cell recycle. Studies were conducted withLactobacillus delbrueckii at a fermentation temperature of 42°C and a pH of 6.25. A cell density of 140 g dry weight/L and a volumetric productivity of 150 g/L.h, with complete glucose consumption, were obtained. It was not possible to obtain a lactic acid concentration above 60 g/L because of product inhibition. A cell purge was not necessary to maintain high viability bacteria culture or to obtain a steady state. At steady state the net cell growth appeared to be negligible. The specific glucose consumption for cell maintenance was 0.33 g glucose/g cells-h.     

Fermentation of sugars in organe peel hydrolysates to ethanol by recombinantEscherichia coli KO11

The conversion of monosaccharides in organe peel hydrolysates to ethanol by recombinantEscherichia coli KO11 has been investigated in pH-controlled batch fermentations at 32 and 37°C. pH values and concentration of peel hydrolysate were varied to determine approximate optimal conditions and limitations of these fermentations. Very high yields of ethanol were achieved by this microorganism at reasonable ethanol concentrations (28–48 g/L). The pH range between 5.8 and 6.2 appears to be optimal. The microorganism can convert all major monosaccharides in organe peel hydrolysates to ethanol and to smaller amounts of acetic and lactic acids. Acetic acid is coproduced in equimolar amounts with ethanol by catabolism of salts of galacturonic acid. South Atlantic Area, Agricultural Research Service, US Department of Agriculture. Mention of a trademark or proprietary product is for identification only, and does not imply a guarantee or warranty of the product by the US Department of Agriculture. All programs and services of the US Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap.     

Production of lactic acid from food wastes

Conversion of food wastes into lactic acid by simultaneous saccharification and fermentation (SSF) was investigated. The process involves saccharification of the starch component in food wastes by a commercial amylolytic enzyme preparation (a mixture of amyloglucosidase, α-amylase, and protease) and fermentation by Lactobacillus delbrueckii. The highest observed overall yield of lactic acid in the SSF was 91% of theoretical. Lactic acid concentration as high as 80 g/L was attainable in 48 h of the SSF. The optimum operating conditions for the maximum productivity were found to be 42°C and pH 6.0. Without supplementation of nitrogen-containing nutrients, the lactic acid yield in the SSF decreased to 60%: 27 g/L of lactic acid from 60 g/L of food waste. The overall performance of the SSF, however, was not significantly affected by the elimination of mineral supplements.     

A new approach to improving the performance ofZymomonas in continuous ethanol fermentations

Although most fermentation ethanol is currently produced in traditional batch processes with yeast, the ethanologenic bacteriumZymomonas mobilis is recognized as an alternative process organism for fuel alcohol production. Different strategies for improving the productivity of ethanol fermentations are reviewed. In batch and open-type continuous fermentations the advantage of replacing yeast byZymomonas relates principally to the 10% higher fermentation efficiency (product yield), whereas in high cell density, closed-type continuous systems (operating with cell recycle or retention) the superior kinetic properties ofZymomonas can be exploited to affect about a five-fold improvement in volumetric productivity. Unlike yeast, the rate of energy supply (conversion of glucose to ethanol) inZymomonas is not strictly regulated by the energy demand and a nongrowing culture exhibits a maintenance energy coefficient that is at least 25 times higher than yeast. As an alternative to process improvement through genetic engineering of the process organism this investigation has taken a biochemical and physiological approach to increasing the kinetic performance ofZ. mobilis through manipulation and control of the chemical environment. Energetically “uncoupled” phenotypes with markedly increased specific rates of ethanol production were generated under conditions of nutritional limitation (nitrogen, phosphate, or potassium) in steady-state continuous culture. The pH was shown to influence energy coupling inZymomonas affecting the maintenance coefficient (m _e ) rather than the max growth yield coefficient (Y _x ^sάx ). Whereas the pH for optimal growth ofZ. mobilis (ATCC 29191) in a complex medium was 6.0–6.5, the specific rate of ethanol production in continuous fermentations was maximal in the range 4.0–4.5. Fermentation conditions are specified for maximizing the specific productivity of aZymomonas-based continuous ethanol fermentation where the potential exists for improving the volumetric productivity in dense culture fermentations with an associated 35–40% reduction in capital costs of fermentation equipment and an estimated savings of 10–15% on cost of product recovery (distillation), and 3–7% on overall production costs based on the projected use of inexpensive feedstocks.     

Enhancement of acid tolerance in Zymomonas mobilis by a proton-buffering peptide

A portion of the cbpA gene from Escherichia coli K-12 encoding a 24 amino acid proton-buffering peptide (Pbp) was cloned via the shuttle vector pJB99 into E. coli JM105 and subsequently into Zymomonas mobilis CP4. Expression of Pbp was confirmed in both JM105 and CP4 by HPLC. Z. mobilis CP4 carrying pJB99-2 (Pbp) exhibited increased acid tolerance (p<0.05) in acidified TSB (HCl [pH 3.0] or acetic acid [pH 3.5]), glycine-HCl buffer (pH 3.0), and sodium acetate-acetic acid buffer (pH 3.5) in comparison to the parent strain (CP4) and CP4 with pJB99 (control plasmid). Although the expression of Pbp influenced survival at a low pH, the minimum growth pH was unaffected. Growth of Z. mobilis in the presence of ampicillin also significantly increased acid tolerance by an unknown mechanism. Results from this study demonstrate that the production of a peptide with a high proportion of basic amino acids can contribute to protection from low pH and weak organic acids such as acetic acid.     

Optimization of l -(+)-lactic acid production using pelletized filamentous Rhizopus oryzae NRRL 395

Lactic acid is used as a food additive for flavor and preservation and a precursor in the development of poly-lactic acid, a product used to make biodegradable plastics and textiles. Rhizopus oryzae NRRL 395 is known to be a strain that produces optically pure l-(+)-lactic acid. The morphology of Rhizopus cultures is complex, forming filamentous, clumps, and pellet mycelia. Different morphology growth has significant effects on lactic acid production. In bioreactors, the filamentous or clump mycelia increase the viscosity of the medium, wrap around impellers, and block the nutrient transportation, leading to a decrease in production efficiency and bioreactor performance. Growing fungi in pellet form can significantly improve these problems. In this study, factors that affect lactic acid production in pelletized flask cultures using R. oryzae NRRL 395 were investigated in detail. Completely randomized designs were used to determine the influence of culture temperature, time, concentration of glucose, and inoculum size. Lactic acid fermentation using clump and pellet morphologies were performed in a 5 L fermentor at the optimal values obtained from flask culture. Finally, fed-batch culture was used to enhance the lactate concentration in broth. The final lactate concentration of fed-batch culture reached 92 g/L. The data presented in the article can provide useful information on optimizing lactic acid production using alternative source materials.     

Corn steep liquor as a cost-effective nutrition adjunct in high-performanceZymomonas ethanol fermentations

The economics of large-scale production of fuel ethanol from biomass and wastes requires the efficient utilization of all the sugars derived from the hydrolysis of the heteropolymeric hemicellulose component of lignocellulosic feedstocks. Glucuronic and 4-0-methyl-glucuronic acids are major side chains in xylans of the grasses and hardwoods that have been targeted as potential feedstocks for the production of cellulosic ethanol. The amount of these acids is similar to that of arabinose, which is now being viewed as another potential substrate in the production of biomass-derived ethanol. This study compared the end-product distribution associated with the fermentation of D-glucose (Glc) and D-glucuronic acid (GlcUA) (as sole carbon and energy sources) byEscherichia coli B (ATCC 11303) and two different ethanologenic recombinants—a strain in whichpet expression was via a multicopy plasmid (pLOI297) and a chromosomally integrated construct, strain KO11. pH-stat batch fermentations were conducted using a modified LB medium with 2% (w/v) Glc or GlcUA with the set-point for pH control at either 6.3 or 7.0. The nontransformed host culture produced only lactic acid from glucose, but fermentation of GlcUA yielded a mixture of ethanol, acetic, and lactic acids, with acetic acid being the predominant end-product. The ethanol yield associated with GlcUA fermentation by both recombinants was similar, but acetic acid was a significant by-product. Increasing the pH from 6.3 to 7.0 increased the rate of glucuronate fermentation, but it also decreased the ethanol mass yield from 0.22 to 0.19 g/g primarily because of an increase in acetic acid production. In all fermentations there was good closure of the carbon mass balance, the exception being the recombinant bearing plasmid pLOI297 that produced an unidentified product from GlcUA. The metabolism of GlcUA by this metabolically engineered construct remains unresolved. The results offered insights into metabolic fluxes and the regulation of pyruvate catabolism in the wild-type and engineered strains. End-product distribution for metabolism of glucuronic acid by the nontransformed, wild-typeE. coli B and recombinant strain KO11 suggests that the enzyme pyruvate-formate lyase is not solely responsible for the production of acetylCoA from pyruvate and that derepressed pyruvate dehydrogenase may play a significant role in the metabolism of GlcUA.     

Continuous fermentation studies with xylose-utilizing recombinant Zymomonas mobilis

This study examined the continuous cofermentation performance characteristics of a dilute-acid “prehydrolysate-adapted” recombinant Zymomonas 39676:pZB4L and builds on the pH-stat batch fermentations with this recombinant that we reported on last year. Substitution of yeast extract by 1% (w/v) corn steep liquor (CSL) (50% solids) and Mg (2 mM) did not alter the coferm entation performance. Using declared assumptions, the cost of using CSL and Mg was estimated to be 12.5c/gal of ethanol with a possibility of 50% cost reduction using fourfold less CSL with 0.1% diammonium phosphate. Because of competition for a common sugar transporter that exhibits a higher affinity for glucose, utilization of glucose was complete whereas xylose was always present in the chemostat effluent. The ethanol yield, based on sugar used, was 94% of theoretical maximum. Altering the sugar ratio of the synthetic dilute acid hardwood prehydrolysate did not appear to significantly change the pattern of xylose utilization. Using a criterion of 80% sugar utilization for determining the maximum dilution rate (D _max), changing the composition of the feed from 4% xylose to 3%, and simultaneously increasing the glucose from 0.8 to 1.8% shifted D _max from 0.07 to 0.08/h. With equal amounts of both sugars (2.5%), D _max was 0.07/h. By comparison to a similar investigation with rec Zm CP4:pZB5 with a 4% equal mixture of xylose and glucose, we observed that at pH 5.0, the D _max was 0.064/h and shifted to 0.084/h at pH 5.75. At a level of 0.4% (w/v) acetic acid in the CSL-based medium with 3% xylose and 1.8% glucose at pH 5.75, the D _max for the adapted recombinant shifted from 0.08 to 0.048/h, and the corresponding maximum volumetric ethanol productivity decreased 45%, from 1.52 to 0.84 g/(L·h). Under these conditions of continuous culture, linear regression of a Pirt plot of the specific rate of sugar utilization vs D showed that 4 g/L of acetic acid did not affect the maximum growth yield (0.030 g dry cell mass/g sugar), but did increase the maintenance coefficient twofold, from 0.46 to 1.0 g of sugar/(g of cell·h).     

Membrane-mediated extractive fermentation for lactic acid production from cellulosic biomass

Lactic acid production from cellulosic biomass by cellulase andLactobacillus delbrueckii was studied in a fermenter-extractor employing a microporous hollow fiber membrane (MHF). This bioreactor system was operated under a fed-batch mode with continuous removal of lactic acid by anin situ extraction. A tertiary amine (Alamine 336) was used as an extractant for lactic acid. The extraction capacity of Alamine 336 is greatly enhanced by addition of alcohol. Long-chain alcohols serve well for this purpose since they are less toxic to micro-organism. Addition of kerosene, a diluent, was necessary to reduce the solvent viscosity. A solvent mixture of 20% Alamine 336, 40% oleyl alcohol, and 40% kerosene was found to be most effective in the extraction of lactic acid. Progressive change of pH from an initial value of 5.0 down to 4.3 has significantly improved the overall performance of the simultaneous saccharification and extractive fermentation over that of constant pH operation. The change of pH was applied to promote cell growth in the early phase, and extraction in the latter phase.     

Mycelial pellet formation by Rhizopus oryzae ATCC 20344

Factors in a cultivation medium affecting fungal growth morphology and funmaric acid production by Rhizopus oryzae ATCC 20344 were investigated. These factors included the initial pH value and trace metals such as zinc, magnesium, iron, and manganese in the cultivation medium. It was found that a significant change in the growth morphology of R. oryzae ATCC 20344 occurs when the initial pH value is varied. A lower initial pH value in the cultivation medium was inhibitory to fungal growth, and fast growth in the cultivation medium at a higher initial pH value promoted, the formation of large pellets or filamentous forms. Trace metals in the cultivation media also had significant effects on pellet formation and fumaric acid fermentation.     

Ethanol production from corn starch in a fluidized-bed bioreactor

The production of ethanol from industrial dry-milled corn starch was studied in a laboratory-scale fluidized-bed bioreactor using immobilized biocatalysts. Saccharification and fermentation were carried out either simultaneously or separately. Simultaneous saccharification and fermentation (SSF) experiments were performed using small, uniform κ-carrageenan beads (1.5–2.5 mm in diameter) of co-immobilized glucoamylase and Zymomonas mobilis. Dextrin feeds obtained by the hydrolysis of 15% drymilled corn starch were pumped through the bioreactor at residence times of 1.5–4h. Single-pass conversion of dextrins ranged from 54–89%, and ethanol concentrations of 23–36 g/L were obtained at volumetric productivities of 9–15 g/L-h. Very low levels of glucose were observed in the reactor, indicating that saccharification was the rate-limiting step. In separate hydrolysis and fermentation (SHF) experiments, dextrin feed solutions of 150–160 g/L were first pumped through an immobilized-glucoamylase packed column. At 55°C and a residence time of 1 h, greater than 95% conversion was obtained, giving product streams of 162–172 g glucose/L. These streams were then pumped through the fluidized-bed bioreactor containing immobilized Z. mobilis. At a residence time of 2 h, 94% conversion and ethanol concentration of 70 g/L were achieved, resulting in an overall process productivity of 23 g/L-h. Atresidence times of 1.5 and 1 h, conversions of 75 and 76%, ethanol concentrations of 49 and 47 g/L, and overall process productivities of 19 and 25 g/L-h, respectively, were achieved.     

Production of lactic acid from pulp mill solid waste and xylose using Lactobacillus delbrueckii (NRRL B445)

Using the simultaneoussaccharification and fermentation (SSF) technique, pulp mill solid waste cellulose was converted into glucose using cellulase enzyme and glucose into lacticacid using NRRL B445. SSF experiments were conducted at various pH levels, temperatures, and nutrient concentrations, and the lactic acid yield ranged from 86 to 97%. The depletion of xylose in SSF was further investigated by inoculating NRRL B445 into a xylose-only medium. On prolonged incubation, depletion of xylose with lactic acid production was observed. An experimental procedure with a nonglucose medium was developed to eliminate the lag phase. From xylose fermentation, Lactobacillus delbrueckii yielded 88–92% lactic acid and 2–12% acetic acid.     

Optimization of seed production for a simultaneous saccharification cofermentation biomass-to-ethanol process using recombinantZymomonas

The five-carbon sugard-xylose is a major component of hemicellulose and accounts for roughly one-third of the carbohydrate content of many lignocellulosic materials. The efficient fermentation of xylose-rich hemicellulose hydrolyzates (prehydrolyzates) represents an opportunity to improve significantly the economics of large-scale fuel ethanol production from lignocellulosic feedstocks. The National Renewable Energy Laboratory (NREL) is currently investigating a simultaneous saccharification and cofermentation (SSCF) process for ethanol production from biomass that uses a dilute-acid pretreatment and a metabolically engineered strain ofZymomonas mobilis that can coferment glucose and xylose. The objective of this study was to establish optimal conditions for cost-effective seed production that are compatible with the SSCF process design. Two-level and three-level full factorial experimental designs were employed to characterize efficiently the growth performance of recombinantZ. mobilis CP4:pZB5 as a function of nutrient level, pH, and acetic acid concentration using a synthetic hardwood hemicellulose hydrolyzate containing 4% (w/v) xylose and 0.8% (w/v) glucose. Fermentations were run batchwise and were pH-controlled at low levels of clarified corn steep liquor (cCSL, 1-2% v/v), which were used as the sole source of nutrients. For the purpose of assessing comparative fermentation performance, seed production was also carried out using a “benchmark” yeast extract-based laboratory medium. Analysis of variance (ANOVA) of experimental results was performed to determine the main effects and possible interactive effects of nutrient (cCSL) level, pH, and acetic acid concentration on the rate of xylose utilization and the extent of cell mass production. Results indicate that the concentration of acetic acid is the most significant limiting factor for the xylose utilization rate and the extent of cell mass production; nutrient level and pH exerted weaker, but statistically significant effects. At pH 6.0, in the absence of acetic acid, the final cell mass concentration was 1.4 g dry cell mass/L (g DCM/L), but decreased to 0.92 and 0.64 g DCM/L in the presence of 0.5 and 1.0% (w/v) acetic acid, respectively. At concentrations of acetic acid of 0.75 (w/v) or lower, fermentation was complete within 1.5 d. In contrast, in the presence of 1.0% (w/v) acetic acid, 25% of the xylose remained after 2 d. At a volumetric supplementation level of 1.5–2.0% (v/v), cCSL proved to be a cost-effective single-source nutritional adjunct that can support growth and fermentation performance at levels comparable to those achieved using the expensive yeast extract-based laboratory reference medium.